Version 2.2 August 2004

Purdue University Cytometry Laboratories

Introduction to WinMDI 2.8 (J. Trotter 1993-1998)

for the Analysis of

Flow Cytometry Listmode Data Files

Objectives of this tutorial:

- Open a listmode data file - Create histograms (single, dual, and 3D histograms) - Perform gating - Perform original analysis - Display statistics

Grald GREGORI, Ph.D. Kathy RAGHEB, M.T.(A.S.C.P.)

J. Paul ROBINSON, Ph.D. Purdue University Cytometry Laboratories

Hansen Life Sciences Research Building, B050 201 S. University Street,West Lafayette, IN 47907-2064,USA

e-mail: gregori@flowcyt.cyto.purdue.edu http://www.cyto.purdue.edu

Version 2.2 August 2004

Contents I. How to Install WinMDI 2.8 .......................................................................................... 1 II. How to Start WinMDI 2.8........................................................................................... 1 III. How to Create a Single-parameter Histogram........................................................ 3 IV. How to Create Histogram Overlay ........................................................................... 5 V. How to Create a Dual-parameter Histogram............................................................ 6

1. How to create a dotplot ............................................................................................... 6 2. How to create a Density plot or a Contour Plot .......................................................... 8 3. How to create a 3-dimensional display..................................................................... 10 4. How to change the parameter labels and font........................................................... 11 5. How to display a title or write annotations on a graph ............................................. 11

VI. Zoom .......................................................................................................................... 12 VII. How to Create and Display Several Graphs on the Screen................................. 13 VIII. Regions and Gates ................................................................................................. 14

1. Create a region on a 1-parameter histogram............................................................. 15 2. Create a region on a 2-parameter histogram............................................................. 16 3. How to create a quadrant .......................................................................................... 17 4. How to delete, move, or adjust a region ................................................................... 18 5. Create gates............................................................................................................... 19

IX. Convert a FCS file to a text file ............................................................................... 20 1. Convert a data file..................................................................................................... 20 2. Only convert the data from a gated region................................................................ 20

X. Display Statistics ........................................................................................................ 21 XI. Work with a Batch File ............................................................................................ 22 XII. Overview .................................................................................................................. 23

What we like ......................................................................................................... 23 What we dislike..................................................................................................... 24

Quick Start in 10 steps.................................................................................................... 25 1. To start WinMDI 2.8................................................................................................. 25 2. To display a 2-parameter histogram ......................................................................... 25 3. To create a 3D plot.................................................................................................... 25 4. To rotate a 3D graph ................................................................................................. 26 5. To display a new single-parameter histogram .......................................................... 26 6. To create a region.................................................................................................. 26 7. To create gates .......................................................................................................... 27 8. To display statistics............................................................................................... 27 9. To create histogram overlay...................................................................................... 28 10. To create 3D view of histograms ............................................................................ 28

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I. How to Install WinMDI 2.8 The Windows Multiple Document Interface software (WinMDI) is freeware

designed by J. Trotter (1993-1998; trotter@scripps.edu) to analyze flow cytometric

listmode data files. You can download it from the URL

http://facs.scripps.edu/software.html or find it on any of the Purdue CD-Roms. To install

WinMDI 2.8 on your computer, just click on the web page link corresponding to your

office system (Windows 95/98/NT or Windows3.1/NT 3.x) and open (or save on your

computer and install later) the corresponding wm28w95.exe or winmdi28.exe programs.

Follow then the instructions.

The minimum system requirements are:

Processor: 486/33 + 387 fpu math chip or better with 8 megabytes memory. Recommended: 486/66 with 16MB RAM or better Monitor: SVGA 800x600 or 1024x768 recommended. OS: MS DOS 5.0 or later + MS Windows 3.1 or later, Windows NT,

Windows XP, or OS/2 Windows 3.1: Use Microsoft's SMARTDrive or other disk cache program.

Hint! I SUGGEST THAT YOU USE LISTMODE DATA FILES WITH 8.3 FORMAT FILE NAMES.

II. How to Start WinMDI 2.8

To start WinMDI just click on the WinMDI icon on the desktop, or use Start

Programs WinMDI WinMDI.exe. The following screen is displayed.

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With WinMDI 2.8 it is possible to create single-parameter histograms, dual-

parameters histograms (also called cytograms), and even 3D histograms. The WinMDI

Display dialog window offers 4 different graph styles (see figures below):

Histogram Dot plot

Density plot Contour plot

- Histogram: displays one or more parameters as a frequency distribution (256

or 1024 channels).

- Dotplot: displays correlated data from any two parameters at a 256x256

resolution. Each dot corresponds to an event analyzed by the flow cytometer.

Of course, several events can occupy the same dot if they have the same

parameter intensities.

- Density plot: displays two parameters as a frequency distribution. Color is

used to code the different frequencies of events.

- Contour plot: like a dotplot, a contour plot displays correlated data from any

two parameters, with contour lines joining points of equal elevation

(frequency distribution).

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III. How to Create a Single-parameter Histogram

From the WinMDI Display dialog window select the Histogram style and click

the OK button. A new window allows you to navigate to the sample folder where the

listmode data file to analyze is recorded.

When the file is selected, press OK. The Select Parameters dialog window appears

for selection of the parameter to display.

Using the scrolling bar, select the parameter you want to display (FS: forward-angle

light scatter; SS: right-angle light scatter; Fl1, Fl2, , Fln: the first, second, and nth

fluorescence recorded by the cytometer). Click the Read button to display the

corresponding histogram.

All the parameters are automatically recorded within each listmode data file. You

do not need to type them in the software.

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Hint! SOMETIMES WINMDI DOES NOT AUTOMATICALLY RECOGNIZE WHICH AMPLIFICATION

IS USED (LINEAR OR LOGARITHMIC). YOU NEED TO SPECIFY THE SCALE BY SELECTING

TOOLS SET LINLOG TO OPEN THE LIN/LOG VARIABLES DIALOG WINDOW. THEN CHECK

THE CORRESPONDING SCALE FOR EACH PARAMETER.

Very often it is necessary to change the position of the histogram and optimize the

size by stretching its different borders and/or corners.

In the Select Parameters dialog window scroll bar, the Show All Parameters

option generates a single graph in which all the histograms of all the different parameters

are plotted together. If you click on the graph and select Format in the popup menu the

Format Histogram dialog window is displayed (see below).

Among the available options you can, for instance:

Set the color of each histogram. Just select the corresponding histogram

name and select the color (scroll menu). You can fill the histograms with

their respective color (check the Solid Fill check box) or leave them

empty (uncheck the box).

Rescale the histograms by changing the Y Scale value.

Change the thickness of the lines (change the Line Width option);

Display all the histograms in black & white (check the B+W check box).

Disp